T cells that restrain immune responses are essential for maintenance of homeostasis and for preventing autoimmune disease. Such regulatory responses prevent local and, very likely, distal site inflammation induced by the intestinal commensal microbiota. Much attention has been lavished on Foxp3+ Treg cells, but other cell types also have key roles in restraining inflammatory processes. One of the key components is the Tr1 cell that produces IL-10, an essential cytokine in systemic homeostasis. Tri cells differentiate in response to IL-27 and TGF-b, and the transcription factors (TFs) AhR and c-Maf are essential for this process both in vitro and in vivo. In the revised grant application, we will undertake a comprehensive analysis of the transcriptional network involved in the differentiation of Tr1 cells in vitro, in vivo in mice, and in human peripheral blood lymphocytes. For the first Specific Aim, we will employ genome-wide ChlP-seq to identify direct targets of Ahr, c-Maf, and other relevant TFs in mouse T cells at different times of polarization towards the Tr1 or control ThO phenotype. The analysis will be performed with CD4+ T cells from wild-type and Ahr- and/or c-Maf-deficient mice to determine if there is cooperativity in binding to target sequences. Genome-wide analysis on cells from wt and mutant mice of histone marks (H3K4, H3K27, and H3K36 methylatlon; H3K9/14 acetylation), Polll and p300 binding, and DNA methylatlon will complement the binding of TFs. Genome-wide RNA sequencing will also be performed on polarized cells from wt and mutant mice at multiple time points. The data will be examined using network algorithms (in collaboration with Richard Bonneau, NYU) to identify other key players in the Tr1 differentiation program. For Specific Aim 2, RNA-seq analysis will be performed on Tri cells obtained from mice after diverse stimuli, including oral gavage with polysaccharide-A from Bacteroides fragilis and injection of anti-CD3 mAb. IL-10-GFP reporter mice will be used for sorting the appropriate cells. Genes modulated both in vitro and in vivo will be the focus of further analysis, including shRNA knockdowns to study the effect on Tr1 differentiation. ChlP-seq and RNA-seq will be performed with cells bearing mutations or RNAi knockdowns of key genes, and the perturbation data will be incorporated in the network analysis. The effect of Ahi- ligands, that modulate IL-10 production, on TF binding and histone modifications will be incorporated in the analysis. For Specific Aim 3, we will validate the role of candidate genes using in vivo models for Tr1 cell differentiation and anti-inflammatory function. This will include mouse models of EAE and transfer colitis. Human Tr1 cells will also be generated in vitro and subjected to treatment with Ahr ligands, and RNA-seq analysis will be performed. Genes identified in the network analysis will guide shRNA knockdown experiments to determine effects on human Tri cell differentiation. We anticipate that these comprehensive analyses and validation studies in vivo in mice and in vitro with primary human T cells will identify novel targets for modulation of human immune responses and for therapeutic applications in multiple sclerosis. In addition. Project 2 will act as a transcriptional core (as a part of multi-tiered core) for the PPG, where we will undertake RNA-seq and ChlPseq analysis for other projects of the PPG.